FI95999C - K2P-prostabilointi - Google Patents

Abstract

PCT No. PCT/EP90/02250 Sec. 371 Date Aug. 2, 1991 Sec. 102(e) Date Aug. 2, 1991 PCT Filed Dec. 19, 1990 PCT Pub. No. WO91/08765 PCT Pub. Date Jun. 27, 1991.Stabilized compositions containing a non-glycosylated human tissue type plasminogen activator derivative are disclosed. The derivative is referred to as K2P pro. The composition has enzymatic activity of at least 1.1 mU/ml, a pH of from 4.5 to 6.5, and contains citrate as well as at least one of a variety of different compounds.

Description

, 95999 K2P prostabilization - K2P prostabilization

Human tissue plasminogen activator (t-PA) is of great therapeutic importance in the breakdown of blood clots, for example in the context of myocardial infarction. t-PA breaks down blood clots by activating plasminogen to plasmin. Plasmin, in turn, dissolves fibrin, the major component of the protein matrix, from coagulated blood.

1 0

Intrinsic t-PA consists of several functional domains F, E, K1, K2 and. Domain P contains a proteolytically active center that causes plasminogen to degrade into plasmin. Genetic engineering of t-PA or various t-PA muteins, some of which have been deleted from regions F, E, K1 and K2, in eukaryotic and prokaryotic cells is known. In this case, t-PA derivatives derived from prokaryotes are synthesized in non-glycosylated form, in contrast to the case of native t-PA.

It is further known that the sugar content has a significant effect on the solubility and aggregation of proteins (J. Biol.

Chem. 263 (1988), 8832-8837). It has now been found that the glycosy-25 unlabeled t-PA mutein with the K2P domain composition is substantially less soluble than the slightly glycosylated · 1 · t-PA derivative. This non-glycosylated t-PA variant Liu only lubricates to a small extent in buffers conventionally intended to solubilize proteins, such as 50 mmol / l Na citrate, pH 6, 50 mmol / l phosphate buffer or physiological NaCl solution. However, when used as a therapeutically active substance, the K2P-enzymatic activity of the non-Glycosylated t-PA derivative should be clearly higher, at least 1.4 MU / ml, preferably 1.4 MU / ml to 10 MU / ml.

It is known from EP-A-0 217 379 to increase the solubility of prokaryotic-derived t-PA (t-PA pro) with neutral or slightly temporal arginine formulations. However, the disadvantage of this method is that the good solubility of t-PA pro is only achieved at very high arginine concentrations. Besides, the pro stability of the highly concentrated t-PA derivative K2P is low under neutral or slightly 5 temporal conditions.

Thus, it is an object of the invention to provide formulations containing the non-glycosylated t-PA derivative K2P pro with an enzymatic activity of at least 1.4 MU / ml, whereby the stability of the t-PA derivative is maintained for a long time.

The object of the invention is solved by a pharmaceutical preparation of a non-glycosylated t-PA derivative K2P pro with an enzymatic activity of at least 1.4 MU / ml and a pH of at least 4.5-6.5, this composition containing and at least one compound derived from the group consisting of a) ascorbic acid, b) EDTA, 20

R ^ N - R - X

amino compounds wherein X is SO 3 H, (CH) NH 2) -CO 2 H, CO 2 H, H, NH 2 or OH, R 1 -C 1 -C 8 alkylene, preferably C 4 -C 7 alkylene, C 3 -C 6 cycloalkylene or benzylidene and R 1 and R 2 are independently H or C 1 - -C3-alkyl, d) of the formula

Y H

(I

H 2 N-C-N-Z

30 guanidine analogs, wherein Y - Η2Ν + or O, z - H or (CH2) mv, (CH2) mCH (NH2) -CO2H, CH (CO2H) - (CH2) mCO2H, V - NH2 or 002Η and 35 m - 1 -4, e) carboxylic acids substituted by one or more hydroxyl, keto and / or other carboxyl groups, f) dimethylbiguanide, g) pyrimidine nucleosides and pyrimidine nucleotides, il · «4» IM »'1 3 95999 h) trehalose, glucosamine, methylgluca.

According to the present invention, K2P is understood to mean a t-PA derivative consisting of ring 2 and protease domains 5 and thus starting from amino acids 174-180 and ending at amino acid 527. K2P pro may additionally contain partially or completely amino acids -3 (Gly) - + 5 (Ile). In this case, a protein starting from amino acid 176 and optionally containing in addition to the amino acids Ser, Tyr, Gln 10 in the range -3-5 is preferred. This description follows the nomenclature given to t-Pa by T.J.R Harris, Protein Engineering, 1st Edition (1987) 449-458. The preparation of such a t-PA derivative K2P pro is described in EP-A-0 382 174. The enzymatic activity of K2P pro is given as a standard unit U according to WHO definition 15. Activity is determined according to H. Lillin, ZGIMAL 42 (1987), 478-486.

The citrate buffer has proven to be particularly suitable for solubilizing K2P pro. The citrate concentration should be, for example, at least 5 mmol / l, preferably 5-100 mmol / l, particularly preferably 50 mmol / l. The pH is adjusted according to the basicity of the compound added, preferably with HCl or a base such as NaOH or KOH.

25

Surprisingly, it was found that the Liu hardness of unglycosylated K2P pro in other buffer systems, for example phosphate buffer, is substantially lower at the same ionic strength at the same pH. It has proved suitable to adjust the pH of the alkaline citrate solutions with HCl, i. that the composition further contains chloride ions. Namely, in the presence of chloride ions, surprisingly, highly concentrated solutions of non-glycosylated K2P pro are substantially more stable than, for example, in the presence of phosphate ions.

The pH of acidic citrate solutions is usually adjusted with NaOH.

A pH of 4.5 to 6.5, particularly preferably a pH of 5 to 6, is suitable for the pharmaceutical preparation according to the invention. Surprisingly, in formulations used for native t-PA with a pH> 7, the pro2 stability of the non-glycosylated t-PA derivative in solution is surprisingly considerable. Thus, the single chain form of K2P pro is stable at pH 7.2 and pH 8 in arginine buffered solutions for only a few days at room temperature or higher. The pharmaceutical preparation according to the invention preferably contains 0.1 to 1 mol / l of ascorbic acid, particularly preferably 0.2 to 0.3 mol / l.

The concentration of EDTA should preferably be 1-200 mmol / l, particularly preferably 10-100 mmol / l.

The amino compounds used in the composition according to the invention are preferably taurine, ε-aminocaproic acid, tranexamic acid, lysine, ornithine, δ-aminovaleric acid, p-aminomethylbenzoic acid, 8-aminooctanoic acid and / or tranexamic acid and / or lysine. Particularly preferred is the use of ε-aminocaproic acid, p-aminomethylbenzoic acid, 7-amino-20-heptanoic acid, tranexamic acid and / or lysine. Preferred concentrations are 0.5-20 mmol, particularly preferred 1-120 mmol.

Equally suitable are 4-aminobutanol-1,5-aminopentanol-1,25-aminohexanol-1,1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diamino- · * pentane, 1,4-diaminobutane and / or 1,3-diaminopropane.

Concentrations of α, ω-diamines and α, ω-amino alcohols suitable for the preparation according to the invention are preferably from 10 to 30,100 mmol / l.

. Taurine and analogous compounds are preferably used in an amount of 0.1 to 0.5 mol / l, very preferably 0.1 to 0.3 mol.

In addition, urea, guanidine butyric acid and / or arginine are preferably used as guanidine analog compounds in the composition according to the invention. The urea concentration is preferably 0.1 to 4 mol / l, particularly preferably 0.5 to 2 mol / l. For other guanidine analog compounds, the concentration is preferably 95-200 mmol / l, particularly preferably 50-100 mmol / l. Suitable carboxylic acids substituted by hydroxy, keto and / or other carboxyl groups are malic acid, lactic acid, fumaric acid and / or 2-oxoglutaric acid. Their concentration is preferably 0.001 to 1 mol / l, preferably 0.01 to 0.5 mol / l.

Dimethylguanidine is used in concentrations of 50-400 mmol / l, particularly preferably 100-300 mol / l.

10

Suitable pyrimidine nucleosides or pyrimidine nucleotides are, for example, thymidine, cytosine and uridine or similar nucleotides. The concentrations used are preferably from 1 to 300 mmol / l, particularly preferably from 10 to 300 mmol / l.

Trehalose, glucosamine and N-methylglucamine are preferably used in concentrations of 1 to 500 mmol / l, particularly preferably 10 to 300 mmol / l.

20

The invention furthermore relates to a composition according to the invention which additionally contains one or more α-aminocarboxylic acids, in particular histidine.

The following are some particularly preferred preparations of the present invention. One formulation contains 50 mmol / l Na-citrate / HCl, pH 6, 2 mol / l urea. The second formulation contains 50 mmol / l Na citrate, pH 6 and 0.5 mol / l to 1 mol / l guanidine. Another 30 formulation contains 50 mmol / l Na citrate, pH 6, and 0.3 mol / l taurine. Another formulation contains 500 mmol / l Na citrate, pH 6, and 0.2 mol / l to 0.3 mol / l ascorbic acid. In addition, another formulation contains 50 mmol / l Na citrate / HCl, pH 35 6, and 300 mmol / l dimethylguanidine. Another formulation contains 50 mmol of Na citrate / HCl, pH 6, and 10-300 mmol of thymidine, uridine or cytosine or 1 00 mmol / l of one of the abovementioned α, ω-diamines or one of the abovementioned α, ω-amino alcohol. Another formulation contains 50 mmol / l Na citrate / HCl, pH 6, and 10-300 mmol of trehalose, glucosamine or N-methylglucamine.

In addition, a particularly preferred formulation according to the invention contains 50 mmol / l Na-citrate / HCl, pH 6, and 1 mmol / l to 10-10 mmol ε-aminocaproic acid, s-aminovaleric acid, 7-aminoheptanoic acid, 8-amino- octanoic acid, p-aminomethylbenzoic acid, L-lysine, ornithine or tranexamic acid. These substances provide an excellent solubility of K2P pro in a surprisingly small molar excess (10-40-fold).

Also preferred are formulations containing 50 mmol / l Na citrate / HCl, pH 6, and a guanidine analog compound, especially arginine and guanidine butyric acid, in a concentration of 50-100 mmol. Another formulation contains 50 mmol / l Na-citrate / HCl, pH 6, and 10-500 mmol of malic acid, lactic acid, fumaric acid or 2-20 oxoglutaric acid. In addition, another formulation contains 50 mmol / l Na citrate, pH 6, and 10-100 mmol / l EDTA.

As can be seen from the examples, combinations of several of the above compounds and citrate also provide good solubility of K2P pro. Suitable are combinations of lysine with arginine, ornithine, glucosamine and / or thymidine or combinations of EDTA with aminocaproic acid, lysine, arginine, glucosamine and / or thymidine. Other combinations of at least two of the above compounds with citrate are also suitable.

·. The invention also relates to a drug based on K2P

pro as an active ingredient, solution or lyophilisate containing the disclosed substances and optionally other pharmaceutically acceptable excipients, auxiliaries, carriers and excipients.

7 95999

The pharmaceutical preparations according to the invention are preferably used as solutions for injection or infusion. This can be done by providing a ready-to-inject solution with a composition according to the invention. There is also a list of possible, pharmaceutical preparations in the form of lyophilizates. These are then reconstituted with injection media or solutions known per se. As the injection medium, water containing conventional additives such as a stabilizer, a solubilizing agent, a buffer and isotonic additives, for example a physiological NaCl concentration, is particularly preferably used as the injection medium. Such additives include, for example, mannitol, tartrate or citrate buffer, ethanol, a complexing agent such as ethylenediaminetetraacetic acid and its non-toxic salts, and high molecular weight polymers such as liquid polyethylene oxide for viscosity control. Carriers for liquid injections should be sterile and are preferably filled into ampoules.

20

In addition, the present invention also encompasses the use of K2P pro in the preparation of the pharmaceutical compositions of the invention.

The following examples illustrate specific embodiments of the invention.

Example 1

Effect of urea on the solubility of non-glycosylated t-PA muteins with the K2P domain composition.

This example illustrates the effect of urea on the solubility of K2P pro. hardness (prepared according to EP-AO 382 174) in citrate-buffered solutions at pH 6.0. As can be seen from Table 1, K2P pro is only sparingly soluble in 50 mM citrate buffer at pH 6.0. By adding urea, the solubility can be greatly improved.

The optimum is 2 moles / 1 urea.

8 95999

Procedure 170 ml of purified K2P pro (dissolved in 0.5 mol / l arginine / H3PC> 4, pH 7.2) is concentrated by ultrafiltration over an Amicon YM 10 membrane. 1 ml of concentrate (activity 5.8 MU / ml) is dialyzed against the buffer shown in Table 1. After centrifugation of the samples, the enzymatic activity in the clear liquid covering the precipitate is measured.

10 Enzymatic activity is given as volume activity in MU / ml and as total activity in MU.

Measurement of K2P activity can be determined in a conventional manner by cleavage of a chromogenic substrate (H. Lill, 15 ZGIMAL 42 (1987), 478-486). Unit U is a unit of t-PA activity according to the WHON National Institute for Biological Standards and Control.

Table 1 20

Buffer Activity

MU / ml MU

50 mmol / l Na-citrate / HCl, pH 6.0 1.03 1.24 25 8 mol / l urea 50 mmol / l Na-citrate / HCl, pH 6.0 2.76 3.59 6 mol / l urea 30 50 mmol / l Na-citrate / HCl, pH 6.0 3.46 4.67 4 mol / l urea 50 mmol / l Na-citrate / HCl, pH 6.0 4.20 5.67 2 mol / 1 urea 35 50 mmol / l Na-citrate / HCl, pH 6.0 2.62 3.27 1 mol / l urea 50 mmol / l Na-citrate / HCl, pH 6.0 0.32 0.93

11; - H; t ISt> Evening: I

9 95999

Example 2

Effect of different substances on the solubility of K2P pro.

This example illustrates the effect of different substances on the solubility of K2P pro in citrate buffered solutions at pH 6. Excellent solubilities (> 2 MU / ml) are achieved with taurine, ascorbic acid, lysine, EACA, glacamic acid, tranexamic acid, dimethylamic acid, dimethyl trehalose, N-methylglucamine, uridine, cytidine, p-aminomethylbenzoic acid, fumaric acid and oxoglutaric acid. In addition, it can be seen that the citrate buffer provides better solubility of the t-PA derivative at the same molar concentration than NH4HCO3, Tris or phosphate buffer.

1 5

Performance:

See example 1.

Concentrate: 20 Activity: 5.8 MU / ml

Table 2

Buffer Activity

25 MU / ml MU

50 mmol / l Na citrate / HCl, pH 6.0 1.92 2.11 50 mmol / l ornithine 50 50 mmol / l Na citrate / NaOH, pH 6.0 2.67 4.00 0.3 mol / 1 taurine ... 50 mmol / l Na-citrate / NaOH, pH 6.0 4.00 4.20 0.3 mol / l ascorbic acid 35 50 mmol / l Na-citrate / HCl, pH 6.0 3, 88 5.80

10 mmol / l EACA

50 mmol / l Na citrate / HCl, pH 6.0 2.6 3.32, 0 95999

10 mmol / l L-lysineM

50 mmol / l Na-citrate / HCl, pH 6.0 5.54 7.36 10 mol / l tranexamic acid 5 50 mmol / l Na-citrate / HCl, pH 6.0 2.96 3.40 0.3 mol / dimethylbiguanide 50 mmol / l Tris / HCl, pH 7.2 0.04 0.06 1 0 50 mmol / l NH4HCO3 0.12 0.19 50 mmol / l Na2HPO4, pH 7.2 0.15 0.23 15 50 mmol / l Na-citrate / HCl, pH 6.0 0.32 0.93 50 mmol / l Na-citrate / HCl, pH 6.0 2.02 2.02 0.3 mol / l glucosamine 20 50 mmol / 1 Na citrate / HCl, pH 6 3.52 3.17 0.3 mol / l trehalose 50 mmol / l Na citrate / HCl, pH 6 1.57 1.88 0.1 mol / l thymidine 25 50 mmol / l Na-citrate / HCl, pH 6 6.32 7.58 0.3 mol / l uridine 50 mmol / l Na-citrate / HCl, pH 6 3.8 4.94 30 mmol / cytosine 50 mmol / l 1 Na citrate / HCl, pH 6 3.46 5.03 10 mmol / l p-aminomethylbenzoic acid 35 50 mmol / l Na citrate / NaOH, pH 6 1, 53 1, 68 0.3 mol / l malic acid 50 mmol / l Na-citrate / NaOH, pH 6 1.59 1.98 l 1 95999 0.3 mol / l lactic acid 50 mmol / l Na-citrate / NaOH, pH 6 4.32 5.16 0.3 mol / 1 fumaa silicic acid 5 50 mmol / l Na-citrate / NaOH, pH 6 4.24 4.66 0.3 mol / l 2-oxoglutaric acid 50 mmol / l Na-citrate / HCl, pH 6 3.36 3.70 10 0, 3 moles / l N-methylglucamine

Example 3 Effect of ε-aminocaproic acid (EACA) on the solubility of K2P pro 1 5

Performance:

See example 1

Concentrate: 20 Activity: 6.3 MU / ml

Table 3

Buffer Activity

25 MU / ml MU

50 mmol / l Na-citrate / HCl, pH 6.0 0.34 0.48 50 mmol / l Na-citrate / HCl, pH 6.0 3.81 4.57

30 10 mmol / l EACA

. 50 mmol / l Na citrate / HCl, pH 6.0 3.52 4.86

· “5 mmol / l EACA

35 50 mmol / l Na citrate / HCl, pH 6.0 2.70 3.83

1 mmol / l EACA

50 mmol / l Na citrate / HCl, pH 6.0 0.58 0.75

0.1 mmol / l EACA

12 95999

Table 3 shows that even an EACA concentration of 1-10 mmol / l (10-40-fold excess compared to K2P pro) provides a significant improvement in solubility in citrate-buffered solutions at pH 6.0.

5

Example 4

Effect of ascorbic acid (EACA) on the solubility of K2P pro

Execution: 10 See example 1

Concentrate:

Activity: 6.3 MU / ml 15 Table 4

Buffer Activity

MU / ml MU

20 50 mmol / l Na citrate / HCl, pH 6.0 0.34 0.48 50 mmol / l Na citrate / NaOH, pH 6.0 4.04 4.24 0.3 mol / l ascorbic acid 25 50 mmol / l Na citrate / NaOH, pH 6.0 2.40 2.93 0.2 mol / l ascorbic acid

The results in Table 4 show that the solubility of K2P pro in citrate buffered solutions is further improved by the addition of ascorbic acid.

Example 5. PH dependence of K2P pro stability in arginine-containing solutions 35

Purified K2P pro is dialyzed against 0.5 M Arg / H3PO4 buffer shown in Table 5 and stored in aliquots at -20, 25 and 37 ° C. After 3, 7, 14 and 21 days, the activity and stimulability of 1 sample in each case were tested by adding fibrin and analyzed by SDS electrophoresis (initial values: activity 1.3 MU / ml; stimulability: 28). The data summarized in Table 5 show that the activity of the samples stored at pH 8 and pH 7.2 has increased significantly by 3 resp. After storage for 7 days at 37 ° C while the stimulability is reduced. At 25 ° C, albeit with a time delay, there is also an increase in activity and a decrease in stimulability. After 14-21 days, a decrease in activity is observed.

Table 5

Activity (MU / Ml) / Stimulability

Time -20 ° C_25 ° C_37 ° C

15 pH 8.0 3 days 1.22 / 36 1.47 / 24 1.45 / 15 7 days 1.23 / 44 1.87 / 15 2.38 / 13 14 days 1.37 / 33 2.08 / 9 1.1 / 10 21 days 1.37 / - 2.14 / 10 1.0 / 7 20 pH 7.2 3 days 1.12 / 39 1.5 / 27 1.7 / 20 7 days 1, 17 / 45 1, 7/21 2.1 / 13 14 days 1, 38/32 1, 7/9 1, 9/9 25 21 days 1.22 / 25 1.7 / 11 1.52 / 8 'pH 6 .0 3 days 0.93 / 34 1.43 / 31 1.54 / 34 7 days 1.05 / 46 1.27 / 42 1.58 / 36 30 14 days 1.23 / 39 1.04 / 20 1 , 45/14 21 days 1.43 / 26 0.95 / 16 1.00 / 8 pH 5.5 3 days 0.85 / 32 1.47 / 40 1.08 / 39 35 7 days 1.4 / 47 1.53 / 68 1.54 / 48 14 days 1.2 / 32 1.3 / 26 1.3 / 24 21 days 1.36 / 26 0.95 / 25 0.95 / 21 1 4 95999 Along with these changes a decrease in the molecular weight of K2P pro is observed (electrophoresis by SDS-PAGE).

Example 6 5 Effect of chloride and phosphate ions on the stability of K2P pro in arginine (Arg) -containing solutions

Purified K2P is pro-dialyzed against the arginine-containing buffer shown below and stored in aliquots at -20, 25, and 37 ° C. After 2, 7 and 14 days, 1 sample each is tested by SDS electrophoresis. It turns out that the single chain form is substantially more stable in the presence of chloride ions than in the presence of phosphate ions. After only 10% to 20% of the sample has decomposed in chloride-containing solutions after storage for 7 days at 37 15 ° C at pH 7.2 and 8, the proportion of decomposed material in the phosphate buffered solutions is 60% to 90%.

Buffer: 0.5 mol / l Arg / H 3 PO 4, pH 8.0 0.5 mol / l Arg / HCl, pH 8.0 0.5 mol / l Arg / H 3 PO 4, pH 7.2 0.5 mol / 1 Arg / HCl, pH 7.2 25 Example 7 Effect of ε-aminocaproic acid on the solubility of K2P pro in citrate and phosphate buffered solutions

Purified K2P pro is concentrated by ultrafiltration to 4.2 30 MU / ml and dialyzed against the buffer shown in Table 8 and centrifuged. After centrifugation of the samples, the activity in the clear supernatant is measured.

35 95999

Table 6 1 5

Buffer Activity MU / ml MU 5 50 mmol / l Na citrate / HCl, pH 6 0.32 0.93 50 mmol / l Na2HPO4 / H3PC> 4, pH 6 0.07 0.10 10 50 mmol / l Na- citrate / HCl, pH 6 3.30 4.60

2 mmol / l EACA

50 mmol / l Na 2 HPO 4 / H 3 PO 4, pH 6 0.90 1/39 2 mmol / l EACA 1 5 50 mmol / l Na citrate / HCl, pH 6 2.43 3.52 2 mmol / l EACA 0.15 mol / 1 NaCl 20 50 mmol / l Na2HPO4 / H3PC> 4, pH 6 1.08 1.57 2 mmol / l EACA 0.15 mol NaCl

Table 6 shows that the solubility of EACA is significantly better in citrate-buffered solutions than in phosphate-buffered solutions.

Example 8

Effect of tranexamic acid (TES) on the solubility of K2P pro

Performance:

See example 1 i> «

Concentrate: 35 Activity: 4.2 MU / ml

Table 7 16 95999

Buffer Activity

MU / ml MU

5 _.__ 50 mmol / l Na citrate / HCl, pH 6 2.66 3.60

10 mmol / l TES

50 mmol / l Na citrate / HCl, pH 6 2.54 3.30

10 5 mmol / l TES

50 mmol / l Na citrate / HCl, pH 6 2.35 3.29

1 mmol / l TES

15 The results show that tranexamic acid (TES) 11 achieves the same solubility of K2P pro as EACA.

1) trans-4-aminomethylcyclohexanecarboxylic acid Example 9 Effect of ω-aminocarboxylic acids on the solubility of K2P pro

Performance:

See Example 1 25

Concentrate: * ’Activity: 4.2 MU / ml

Table 8 30

Buffer Activity

MU / ml MU

50 mmol / l Na-citrate / HCl, pH 6 0.32 0.93 35 50 mmol / l Na-citrate / HCl, pH 6 3.12 4.05 10 mmol / l 8-aminooctanoic acid 50 mmol / l Na citrate / HCl, pH 6 3.54 4.42 1 7 95999 10 mmol / l 7-aminoheptanoic acid 50 mmol / l Na citrate / HCl, pH 6 4.22 2.90 10 mmol / l EACA 5 50 mmol / 1 Na citrate / HCl, pH 6 1.83 2.47 10 mmol / l <5-aminovaleric acid

Example 10 Effect of guanidine analogs on the solubility of K2P pro

Performance:

See Example 1 15 Concentrate:

Activity: 4.2 MU / ml

Table 9 20 Buffer Activity

MU / ml MU

50 mmol / l Na-citrate / HCl, pH 6 1.42 2.06 50 mmol / l arginine 25 50 mmol / l Na-citrate / HCl, pH 6 2.21 3.08 ·· 5 mmol / l guanidine butyric acid

The results show that the guanidine group clearly improves the solubility of the K2P project.

Example 11 v Effect of EDTA on the solubility behavior of K2P pro 35 Procedure:

See example 1

Concentrate:

Activity: 4.2 MU / ml is 95999

Table 10

Buffer Activity

MU / ml MU

5 ____ 50 mmol / l EDTA / NaOH, pH 6 0.22 0.03 0.3 mol / l EDTA / NaCH, pH 6 1.95 1.95 50 mmol / l Na citrate / NaOH, pH 6 1, 80 2.25

50 mmol / l EDTA

10 50 mmol / l Na citrate / NaOH, pH 6 3.36 4.03

100 mmol / l EDTA

50 mmol / l Na citrate / HCl, pH 6 0.32 0.93 1 5

Table 10 shows that the combination of EDTA and citrate had more than an additive effect on the solubility of K2P pro.

20 Example 12

Effect of amino acids alone or in combination on the solubility of K2P pro

Execution: 25 See Example 1

Concentrate:

Activity: 4.2 MU / ml 30 Table 11

Buffer Activity

MU / ml MU

35 50 mmol / l Na-citrate / HCl, pH 6 1.42 2.06 50 mmol / l arginine 50 mmol / l Na-citrate / HCl, pH 6 2.81 3.94 10 mmol / l L-lysine 1 9 95999 50 mmol / l Na-citrate / HCl, pH 6 2.00 2.6 1 mmol / l L-lysine 50 mmol / l Na-citrate / HCl, pH 6 2.56 3.84 5 10 mmol / l L-lysine 10 mmol / l arginine 50 mmol / l Na-citrate / HCl, pH 6 3.27 4.9 10 mmol / l lysine 10 50 mmol / l arginine 50 mmol / l Na-citrate / HCl, pH 6 3 .18 4.13 10 mmol / l lysine 10 mmol / l arginine 15 10 mmol ornithine 50 mmol / l Na citrate / HCl, pH 6 2.70 3.78 10 mmol / l lysine 50 mmol / l arginine 20 10 mmol ornithine 50 mmol / l Na citrate / HCl, pH 6 3.34 4.30 10 mmol / l lysine 50 mmol / l arginine 25 50 mmol ornithine Example 13 Effect of ω-amino alcohols on the solubility of K2P pro 30 Procedure:

See example 1

Concentrate:

Activity: 4.9 MU / ml 35

Table 12 20 9 5 9 9 9

Buffer Activity

MU / ml MU

5 - 50 ramols / l Na citrate / HCl, pH 6 1.84 2.4 50 mmol / l 4-aminobutanol-1 50 mmol / l Na citrate / HCl, pH 6 2.54 3.55 10 50 mmol / 1 5-aminopentanol-1

Example 14

Effect of different combinations of substances on the solubility of K2P pro 1 5

Performance:

See example 1

Concentrate: 20 Activity: 5.5 MU / ml

Table 13

Buffer Activity

25 MU / ml MU

: * 50 mmol / l Na citrate, pH 6 4.24 5.08 pH

50 mmol / l EDTA 1 mmol / l EACA

30 50 mmol / l glucosamine 50 mmol / l Na citrate, pH 6 3.63 5.08

10 mmol / l EDTA 1 mmol / l EACA

35 10 mmol / l glucosamine 50 mmol / l Na citrate, pH 6 3.70 5.14

10 mmol / l EDTA 1 mmol / l EACA

il Μ'Ί Hedgehog H-l a; 21 95999 10 mmol / l glucosamine 50 mmol thymidine 50 mmol / l Na citrate, pH 6 4.00 5.40

5 50 mmol / l EDTA

1 mmol / l lysine 50 mmol / l glucosamine 50 mmol / l Na citrate, pH 6 3.04 3.95

10 50 mmol / l EDTA

1 mmol / l lysine 10 mmol / l glucosamine 50 mmol / l Na citrate, pH 6 3.12 4.52

15 10 mmol / l EDTA

1 mmol / l lysine 10 mmol / l glucosamine 10 mmol thymidine 20 50 mmol / l Na citrate, pH 6 2.60 3.77 10 mmol / l EDTA 1 mmol / l lysine 10 mmol arginine 10 mmol / l glucosamine 25 10 mmol / l thymidine: 50 mmol / l Na citrate, pH 6 2.94 4.12 10 mmol / l EDTA 1 mmol / l lysine 30 25 mmol arginine 10 mmol / l glucosamine 10 mmol / l thymidine 50 mmol / l Na citrate, pH 6 2.80 3.64 35 1 mmol / l lysine 10 mmol arginine 10 mmol / l glucosamine 10 mmol / l thymidine 22 95999 50 ramol / l Na citrate, pH 6 3.20 4.32 10 mmol / 1 EDTA 1 mmol / l lysine 25 mmol arginine 5 10 mmol / l glucosamine 25 mmol / l thymidine 50 mmol / l Na citrate, pH 6 4.56 5.90 10 mmol / l EDTA 10 1 mmol / l lysine 25 mmol mmol arginine 10 mmol / l glucosamine 50 mmol / l thymidine 15 50 mmol / l Na citrate, pH 6 3.24 4.54 1 mmol / l lysine 25 mmol / l arginine 10 mmol / l glucosamine 50 mmol / l thymidine idine 20 50 mmol / l Na citrate, pH 6 3.54 4.95 1 mmol / l lysine 25 mmol / arginine 10 mmol / l glucosamine 25 25 mmol / l thymidine 50 mmol / l Na citrate, pH 6 3, 60 4.68 1 mmol / l lysine 25 mmol / l arginine 30 50 mmol / l glucosamine 25 mmol / l thymidine 50 mmol / l Na citrate, pH 6 5.85 5.85 1 mmol / l lysine 35 25 mmol / l 1 arginine 100 mmol / l glucosamine 25 mmol / l thymidine il UL.t Mii i I ill! 23 9 5 9 9 9

Example 15 Effect of α / ω-diamines on the solubility of K2P pro

Execution: 5 See example 1

Concentrate:

Activity: 4.9 MU / ml 10 Table 14

Buffer Activity

MU / ml MU

15 50 mmol / l Na-citrate / HCl, pH 6 3.12 4.05 50 mmol / l 1,9-diaminononane 50 mmol / l Na-citrate / HCl, pH 6 2.80 3.64 50 mmol / l 1,8-diaminooctane 20 50 mmol / l Na-citrate / HCl, pH 6 3.42 4.45 50 mmol / l 1,6-diaminohexane 50 mmol / l Na-citrate / HCl, pH 6 3.52 4.40 25 50 mmol / l 1,5-diaminopentane: 50 mmol / l Na-citrate / HCl, pH 6 3.96 5.15 50 mmol / l 1,4-diaminobutane 30 50 mmol / l Na-citrate / HCl, pH 6 3.33 4.23 50 mmol / l 1,3-diaminopropane

Claims (23)

95999 24 A process for the preparation of a non-glycosylated t-PA derivative consisting of the Kringle 2 and protease domains starting at one of amino acids 174-180 and ending at amino acid 527 and which may additionally contain all or part of the amino acids -3 (Gly) - + 5 (Ile) ( K2P pro), having an enzymatic activity of at least 1.4 MU / ml and a pH of at least 4.5-6.5, for the preparation of a pharmaceutical preparation, characterized in that the t-PA derivative K2P pro is dissolved in citrate and at least one -10 vista compounds with: a) ascorbic acid, b) EDTA, c) an amino compound of formula rjr2N - R - X 15 wherein X = SO 3 H, CH (NH 2) -CO 2 H, CO 2 H, H, NH 2 or OH, R = C 1 -C 9 -alkylene, preferably C4-C7-alkylene, C3-C6-cycloalkylene or benzylidene and R1 and R2 are independently H or C1-C3-alkyl, d) 20. H II I H2N-C-N-Z guanidine analog compound, wherein Y is H2N + or 0, Z = H or (CH2) mV, (CH2) mCH (NH2) -CO2H, CH (CO2H) - (CH2) mCO2H,: V = NH2 or CO2H and m = 1-4, e) one or more hydroxyl- , keto acids substituted by keto and / or other carboxyl groups, f) dimethyl biguanide,. g) pyrimidine nucleosides and pyrimidine nucleotides, • h) trehalose, glucosamine, N-methylglucamine, and the resulting solution is brought into a suitable pharmaceutical form. 35 Process according to Claim 1, characterized in that the amino compound is taurine, ε-aminocaproic acid, tranexamic acid, lysine, ornithine, δ-aminovaleric acid, p-aminomethylbenzoic acid, 4-aminobutanol-1,5-aminopentanol-1,6 - 11:. HIM II 1 M - - f 25 95999 aminohexanol-1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane , 1,3-diaminopropane, 8-aminooctanoic acid and / or 7-aminoheptanoic acid. 5 Process according to Claim 1, characterized in that the guanidine analogue compound is urea, guanidine butyric acid and / or arginine. Process according to Claim 1, characterized in that the substituted carboxylic acid is malic acid, lactic acid, fumaric acid and / or 2-oxoglutaric acid. Process according to Claim 1, characterized in that one or more α-aminocarboxylic acids, preferably histidine, are additionally added. Process according to one of Claims 1 to 5, characterized in that the concentration of citrate is 5 to 100 mmol / l, preferably 50 mmol. Process according to one of Claims 1 to 6, characterized in that chloride ions are additionally added. Process according to Claim 1, characterized in that 50 mmol / l of Na citrate, pH 6, and 0.1 to 1 mol / l, preferably 0.2 to 0.3 mol / l, of ascorbic acid are used. Process according to Claim 1, characterized in that 50 mmol / l Na citrate, pH 6, and 1 to 200 mmol / l, preferably 10 to 100 mmol / l, EDTA are used. « Process according to Claim 1, characterized in that 50 mmol / l Na citrate, pH 6, and 0.1-0.5 35 mol / l, preferably 0.1-0.3 mol / l, taurine are used. II. Process according to Claim 1, characterized in that 50 mmol / l Na-citrate / HCl, pH 6, and 0.5-20 95999 26 mmol / l, preferably 1-10 mmol / l, ε-aminocaproic acid, δ-aminovaleric acid are used , lysine, ornithine, tranexamic acid, p-aminomethylbenzoic acid, 7-aminoheptanoic acid and / or 8-aminooctanoic acid. 5 Process according to Claim 1, characterized in that 50 mmol / l Na-citrate / HCl, pH 6, and 10-100 mmol / l of 4-aminobutanol-1,5-aminopentanol-1,6-aminohexanol 1,1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane or 1,3-diaminopropane. Process according to Claim 1, characterized in that 50 mmol / l Na citrate / HCl, pH 6, and 0.1 to 4 mol / l, preferably 0.5 to 2 mol, of urea are used. Process according to Claim 1, characterized in that 50 mmol / l Na citrate / HCl, pH 6, and 10-200 mmol / l, preferably 50 to 100 mmol, guanidine butyric acid or arginine are used. Process according to Claim 1, characterized in that 50 mmol / l of Na citrate, pH 6, and 0.001 to 1 mol / l, preferably 0.01 to 0.5 mol, of malic acid, lactic acid, fumaric acid or 2 -oksoglutaarihappoa. Process according to Claim 1, characterized in that 50 mmol / l Na citrate / HCl, pH 6, and 50-400 mmol / l, preferably 100-300 mmol, of dimethyl biguanide are used. 30 Process according to Claim 1, characterized in that 50 mmol / l Na citrate / HCl, pH 6, and 1 to 300 mmol / l, preferably 10 to 300 mmol / l thymidine, cytosine or uridine are used. 35 Process according to Claim 1, characterized in that 50 mmol / l Na citrate / HCl, pH 6 and 1-500 27 95999 mmol / l, preferably 10-300 mmol, trehalose, glucosamine or N-methylglucamine are used. . Process according to Claim 1, characterized in that a combination of 50 mmol / l Na citrate and compounds from the group a) to h) according to Claim 1 is used. Process according to one of Claims 1 to 19, characterized in that other conventional pharmaceutical additives, auxiliaries and / or carriers are additionally added thereto. A method according to claim 1, characterized in that the pharmaceutical administration form is a solution for injection or a lyophilisate. 15